A unifying theme of this project and the program project is that specification of cardiac lineages and expression of cardiac restricted genes requires combinatorial interactions between core transcription factors found to be enriched during the emergence of cardiac progenitor cells and the modular organization of regulatory regions of these genes to direct cardiac and chamber specific gene activity. One of these core factors is Nkx2-5, a homeodomain gene, which is essential for normal heart morphogenesis, myogenesis and myocyte differentiation. Developmental regulation of the murine Nkx2-5 genetic locus is highly complex and modular. Currently, the left ventricular and atrium enhancers are still not identified. We propose to establish an efficient in vivo system for enhancer mapping of the mouse Nkx2-5 gene using Nkx2-5 BAC transgenics. Using a combination of strategies, that include nested transposon 10 deletions, identification of conserved genomic sequence alignments, transgenics and knockouts, we propose to detect and characterize potential chamber specific enhancer/repressor regions of Nkx2-5. Another core factor, serum response factor (SRF) may play a leading role in the commitment of cardiac progenitors by virtue of its obligatory requirement for cardiac mesoderm formation and by its ability for interacting in a combinatorial manner with other early cardiac enriched transcription factors. SRF assists as a docking surface for the binding of cofactors, such as Nkx2-5 and GATA-4 that may confer the regulation of specific gene programs. We have found that restricted expression of SRF closely overlapped with such as Nkx2-5, GATA4 and CRPI/2 (smooth muscle Lim only factors), in early vertebrate embryos, coincident with the earliest appearance of smooth muscle target genes and nascent myocardial cells. This transcription factor complex,was central for the de novo upregulation of smooth muscle specified genes. We discovered that the appearance of dHAND in combination with Nkx2-5, SRF, GATA4, and CRPt/2 activated striated beta-actin gene activity. We propose to investigate the molecular basis underlying the specification, and maintenance of cardiac muscle cell differentiation, and the modular regulation of Nkx2-5 to understand early development of multichambered heart and provide opportunities for cell replacement therapy and heart regeneration. The following aims are: To determine how modular genetic regions provide for cardiogenic regulatory activity of the Nkx2-5 gene locus? To determine how SRF specifies procardiac mesoderm to committed cardiogenic cell types? To determine how CRP Lim only family members direct early procardiogenic gene activity and how the appearance of dHAND and CRP3 switches on cardiogenic gene activity in committed cardiomyoctyes?